Foxp3+ Tregs are a unique subset of CD4+ T cells responsible for self-tolerance and for the prevention of autoimmune disease (Shevach E M, Immunity, 2009; 30(5):636-645). Adoptive Treg infusion has been suggested as a potential therapy for the prevention of Graft versus Host Disease (GVHD) following stem cell transplantation, organ allograft rejection, and for the treatment of autoimmune diseases such as type I diabetes and multiple sclerosis (Roncarolo M-G, Battaglia M., Nat Rev Immuno., 2007; 7(8):585-598; Riley J L, June C H, Blazar B R, Immunity, 2009; 30(5):656-665). Adoptive transfer of Foxp3+ Tregs in mouse models has been shown to prevent acute and chronic GVHD without negative effects on the graft versus leukemia response (Hoffmann P, Ermann J, Edinger M, Fathman C G, Strober S, J Exp Med, 2002; 196(3):389-399). More recently, a number of groups have reported that co-transfer of expanded Tregs from umbilical cord samples (Brunstein C G, Miller J S, Cao Q, et al., Blood, 2011; 117(3):1061-1070) or from peripheral blood appears to be both safe (Trzonkowski P, Bieniaszewska M, Jukinska J, et al., Clin Immunol, 2009; 133(1):22-26) and in one study remarkably effective in preventing acute GVHD following stem cell transplantation (Di Ianni M, Falzetti F, Carotti A, et al., Blood, 2011; 117(14):3921-3928).
Although considerable enthusiasm has been generated for adoptive Treg therapy, several major issues remain to be resolved. First, most clinical applications of Treg therapy will require large numbers of cells and optimal methods for Treg expansion are now being explored. Expansion of highly purified populations of human Tregs also frequently results in loss of Foxp3 expression during the expansion process. Secondly, in contrast to studies in the mouse, Foxp3 expression can be readily induced during in vitro stimulation of conventional human T cells (Shevach E M, Tran D Q, Davidson T S, Andersson J, Eur J Immunol, 2008; 38(4):915-917). T cells induced in vitro to express Foxp3 frequently lack a Treg phenotype, continue to make effector cytokines and lack in vitro suppressive function (Shevach E M, Tran D Q, Davidson T S, Andersson J, Eur J Immunol, 2008; 38(4):915-917). Thus, expression of Foxp3 cannot be considered a completely reliable marker for functional human Tregs.
A number of approaches have been used to address these problems. Combined use of several surface markers (CD127lo and CD25hi) has facilitated isolation of more highly enriched populations of Foxp3+ T cells with less contamination by CD25int activated T cells (Liu W, Putnam A L, Xu-Yu Z, et al., J Exp Med. 2006; 203(7):1701-1711). Addition of inhibitors of the mTOR pathway, such as rapamycin, block the expansion of contaminating conventional T cells and favor the expansion of Tregs, but purity greater than 60% is rarely achieved after several rounds of expansion depending on the starting population (Hippen K L, Merkel S C, Schirm D K, et al., American Journal of Transplantation, 2011; 11(6): 1148-1157). CD4+CD25+CD45RA+Foxp3+ T cells, although a minor subpopulation (5-30%) of the Foxp3+ pool in adults, appear to have a greater propensity to expand in culture and have enhanced stability of Foxp3 expression compared to CD4+CD25+CD45RO+Foxp3+ T cells (Miyara M, Yoshioka Y, Kitoh A, et al., Immunity, 2009; 30(6):899-911).
Foxp3+ Tregs can be divided into two potentially distinct subpopulations. One population is generated in the thymus and has been termed natural (n)Tregs. A second population is generated extrathymically in peripheral sites and has been termed induced (i) Tregs or adaptive Treg. It has recently (Thornton A M, Korty P E, Tran D Q, et al., J. Immunol. 2010; 184(7):3433-3441) been demonstrated that the transcription factor, Helios, a member of the Ikaros gene superfamily, is expressed in 70% of both mouse and human Foxp3+ T cells. Foxp3+ Helios− T cells are primarily iTregs as Foxp3+ T cells induced in vitro are Helios−, and Foxp3+ T cells induced in vivo in response to oral antigen administration, antigen administered i.v., or T cells activated in response to lymphopenia are almost exclusively Helios−.
Currently, there is no reliable method for producing populations of functional, human Tregs that can be used for treating disease. The present invention provides such a method.